Method of measuring creatine kinase activity

Abstract

A method and composition for use therein for measuring the activity of creatine kinase (hereinafter referred to as "CK") having good storage stability is disclosed. More particularly, a composition for measurement of CK activity which comprises as main components phosphoglycerate kinase (hereinafter referred to a "PGK") which catalyzes the following reaction 2 and glyceraldehyde-3-phosphate dehydrogenase (hereinafter referred to as "GAPDH") which catalyzes the following reaction 3. Reaction 1 is catalyzed by the creatine kinase. Reaction 1 Creatinephosphate+Adenosine-5'-diphosphate (hereinafter referred to as "ADP")-><-Creatine+Adenosine-5'-triphosphate (hereinafter referred to as "ATP") Reaction 2 ATP+3-Phosphoglycerate-><-ADP+1,3-Diphosphoglycerate Reaction 3 1,3-Diphosphoglycerate+Reduce beta -nicotinamideadenine dinucleotide (hereinafter referred to as "NADH")-><-Glyceraldehyde-3-phosphate+ beta -nicotinamideadenine dinucleotide (hereinaf

What is claimed is: 
     
       1. A method for measuring creatine kinase (CK) activity which comprises combining a test sample with the reagents necessary to carry out the following reactions at a pH of about 6 to 7.5:   Creatine phosphate+ADP⃡Creatine+ATP            (1)       ATP+3-Phosphoglycerate⃡ADP+1,3-Diphosphoglycerate (2)       1,3-[Phosphoglycerate]Diphosphoglycerate+NADH⃡Glyceraldehyde-3-phosphate+NAD+Inorganic phosphate                          (3)     wherein one of those reagents is a composition consisting essentially of 0.5 to 50 u/ml of phosphoglycerate kinase (PGK) and 0.5 to 100 u/ml of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the ratio of PGK to GAPDH being about 1:0.2 to 2 and the total amount of PGK and GAPDH in the test being about 0.1 to 30 mg/ml, and as another one of said reagents a buffer solution capable of maintaining said pH of about 6 to 7.5, and optically measuring the rate of reduction of NADH optical absorption.   
     
     
       2. The process of claim 1, wherein said NADH absorption is measured at a wavelength of 334 nm, 340 nm or 366 nm. 
     
     
       3. A method for measuring creatine kinase (CK) activity which comprises combining a test sample with a reagent composition necessary to carry out the following reactions:   Creatine phosphate+ADP⃡Creatine+ATP            (1)       ATP+3-Phosphoglycerate⃡ADP+1,3-Diphosphoglycerate (2)       1,3-Diphosphoglycerate+NADH⃡Glyceraldehyde-3-phosphate+NAD+Inorganic phosphate                                            (3)     wherein the reagent composition for carrying out said reactions consists essentially of:     ______________________________________                                    
PGK                    0.5-50 μ/ml                                     
GAPDH                  0.5-100 μ/ml                                    
LDH                    0-250 μ/ml                                      
ADP                    0.5-10 mM                                          
Creatine phosphate     10-50 mM                                           
Magnesium containing salt                                                 
                       5-50 mM                                            
3-Phosphoglycerate     5-50 mM                                            
Sulfhydryl compounds   0.1-100 mM                                         
Chelating agents       0-10 mM                                            
Polyhydroxy compounds  0-0.2 mM                                           
AMP                    0-25 mM                                            
Diadenosine-pentaphosphate                                                
                       0-100 μM                                        
Sterilizers            0-10 mM                                            
Buffer (to maintain a                                                     
pH: 6 to 7.5)          0.05-0.5 M                                         
NADH                   0.1-0.5 mM                                         
Alkaline carbonate or                                                     
alkaline bicarbonate   0-10 mM                                            
Volume fraction of serum in                                               
the assay mixture      0.01-0.5,                                          
______________________________________                                    
     the ratio of PGK to GAPDH being about 1:0.2 to 2 and the total amount of PGK and GAPDH in the test being about 0.1 to 30 mg/ml, and optically measuring the rate of reduction in NADH optical absorption.   
     
     
       4. A method for measuring creatine kinase (CK) activity which comprises combining a test sample with the reagents necessary to carry out the following reactions at a pH of about 6 to 7.5:   Creatine phosphate+ADP⃡Creatine+ATP            (1)       ATP+3-Phosphoglycerate⃡ADP+1,3-Diphosphoglycerate (2)       1,3-Diphosphoglycerate+NADH⃡Glyceraldehyde-3-phosphate+NAD+Inorganic phosphate                                            (3)     wherein one of those reagents is a composition consisting essentially of 0.5 to 50 u/ml of phosphoglycerate kinase (PGK), 0.5 to 100 u/ml of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the ratio of PGK to GAPDH being about 1:0.2 to 2 and the total amount of PGK and GAPDH in the test being about 0.1 to 30 mg/ml, and as another one of said reagents a buffer solution capable of maintaining said pH of about 6 to 7.5, and at least one of a stabilizer, a chelating agent, a sterilizer and lactate dehydrogenase (LDH), and optically measuring the rate of reduction of NADH optical absorption.